The mosquito-borne dengue (DEN) viruses, members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome. A single polypeptide is co-translationally processed by viral and cellular proteases generating three structural proteins (C, M, and E) and at least seven non-structural proteins. The genome organization of the DEN viruses is 5-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3 (UTR untranslated region, C capsid, prM membrane precursor, E envelope, NS nonstructural). There are four dengue virus serotypes (DEN1, DEN2, DEN3, and DEN4) that circulate in tropical and subtropical regions of the world inhabited by more than 2.5 billion people. Annually, there are an estimated 50-100 million dengue infections and hundreds of thousands of cases of the more severe and potentially lethal dengue hemorrhagic fever/shock syndrome (DHF/DSS) with children bearing much of the disease burden. DEN viruses are endemic in at least 100 countries and cause more human disease than any other mosquito-borne virus. In at least eight Asian countries, the DEN viruses are a leading cause of hospitalization and death in children. Unfortunately, many countries affected by DEN viruses have very limited financial resources for healthcare, and the economic burden of DEN disease is considerable. As such, an economical vaccine that prevents disease caused by the DEN viruses is a global public health priority. ? ? The cost-effectiveness, safety, long-term immunity, and efficacy associated with the live attenuated vaccine against yellow fever virus, another mosquito-borne flavivirus, serves as a model for the feasibility of a live attenuated DEN virus vaccine. However, the development of a live attenuated dengue vaccine has been complicated by several factors. First, it has been difficult to develop monovalent vaccines against each of the four serotypes that exhibit a satisfactory balance between attenuation and immunogenicity. Second, an effective live attenuated dengue virus vaccine must consist of a tetravalent formulation of components representing each serotype because multiple serotypes typically co-circulate in a region, each DEN serotype is capable of causing disease, and the introduction of additional serotypes is common. In addition, the association of increased disease severity (DHFDSS) in previously infected persons undergoing an infection by a different dengue serotype necessitates a vaccine that will confer long-term protection against all four serotypes. Third, it has been difficult to formulate a tetravalent vaccine with low reactogenicity that induces a broad neutralizing antibody response against each DEN serotype. Fourth, a dengue vaccine must confer protection against a wide range of genetically diverse subtypes that are dispersed around the world and can be readily introduced into a new region by international travel. Fifth, a dengue vaccine must be produced economically so that it can be made available to populations that need it most. ? ? We have tried to address these issues as part of a program to generate a live attenuated tetravalent dengue virus vaccine. To maximize the likelihood that suitable vaccine candidates would be identified, monovalent vaccine candidates for DEN1-4 were generated by two distinct recombinant methods and found to be attenuated and immunogenic in mouse and rhesus monkey models. In one method, deletion of 30 contiguous nucleotides from the 3 UTR of wild type cDNA clones of DEN1-4 was used to generate vaccine candidates. Specifically, the deletion of nucleotides 10478-10507 of the 3 UTR (del30) of recombinant wild type DEN4 yielded a vaccine candidate, rDEN4del30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans (9). Incorporation of the del30 mutation into infectious cDNA clones of DEN1, but not DEN2 or DEN3, at a site homologous to that in DEN4 attenuated these viruses for rhesus monkeys. Using a second method, antigenic chimeric viruses were generated by replacing wild type M and E structural genes of rDEN4del30 with those from DEN2 or DEN3, and the resulting chimeric viruses were attenuated and immunogenic in rhesus monkeys. The dengue virus vaccine program is predominantly in a clinical mode at this time. The preclinical phase of this program is currently devoted largely to support the manufacture of clinical lots of vaccines suitable for study in volunteers. Multiple clinical lots of dengue 1-4 have been manufactured over the past year, focusing largely on the DEN3 vaccine candidates. ? ? Dengue serotype 1 vaccine development? ? The attenuation phenotype of rDEN1/4del30(ME) in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4del30(ME) should be considered for evaluation in a clinical trial. Further research with this promising vaccine candidate is on hold pending outcome of ongoing clinical studies with the lead rDEN1del30 candidate that continues to look good in clinical trials.? ? Dengue serotype 3 vaccine development? ? The dengue virus type 3 (DEN3) vaccine candidate, rDEN3del30, was previously found to be under-attenuated in both SCID-HuH-7 mice and rhesus monkeys and to have decreased infectivity for humans. Herein, two strategies have been employed to generate attenuated rDEN3 vaccine candidates that retain the full complement of structural and nonstructural proteins of DEN3 and thus are able to induce humoral or cellular immunity to each of the DEN3 proteins. First, using the predicted secondary structure of the DEN3 3 untranslated region (3-UTR), nine novel deletion mutant viruses were designed and found to be viable. Four of nine deletion mutants replicated efficiently in Vero cells and were genetically stable. Second, chimeric rDEN3 viruses were generated by replacement of the 3-UTR of the rDEN3 cDNA clone with that of rDEN4 or rDEN4del30 yielding the rDEN3-3D4 and rDEN3-3D4del30 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN3del30/31 and rDEN3del86, or with rDEN3-3D4del30 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DEN3 challenge. The rDEN3del30/31 virus showed a strong host range restriction phenotype with complete loss of replication in C6/36 mosquito cells despite robust replication in Vero cells. In addition, rDEN3del30/31 had reduced replication in Toxorynchites mosquitoes following intrathoracic inoculation. Clinical lots of the rDEN3del30/31 and rDEN3-3D4del30 have been manufactured, and INDs have been submitted or are about to be submitted. Phase 1 clinical trials with each should begin within four months.? ? An additional DEN3/4del30 vaccine candidate, in which the prM and E genes come from the Sri Lanka strain of DEN3 virus rather than the Slemens strain that had reduced infectivity for humans, has been generated and is being tested for infectivity and immunogenicity in rhesus monkeys. It is hoped that the clinically more prM and E genes from the more virulent Sri Lanka virus will enhance the infectivity of the virus for humans.

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Blaney Jr, Joseph E; Sathe, Neeraj S; Hanson, Christopher T et al. (2007) Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the structural genes of rDEN4 and rDEN4Delta30 with those of DEN1. Virol J 4:23
Whitehead, Stephen S; Blaney, Joseph E; Durbin, Anna P et al. (2007) Prospects for a dengue virus vaccine. Nat Rev Microbiol 5:518-28
Blaney Jr, Joseph E; Durbin, Anna P; Murphy, Brian R et al. (2006) Development of a live attenuated dengue virus vaccine using reverse genetics. Viral Immunol 19:10-32
Blaney Jr, Joseph E; Matro, Jennifer M; Murphy, Brian R et al. (2005) Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys. J Virol 79:5516-28
Blaney Jr, Joseph E; Hanson, Christopher T; Hanley, Kathryn A et al. (2004) Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2. BMC Infect Dis 4:39
Hanley, Kathryn A; Manlucu, Luella R; Manipon, Gracielle G et al. (2004) Introduction of mutations into the non-structural genes or 3' untranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity. Vaccine 22:3440-8
Blaney Jr, Joseph E; Hanson, Christopher T; Firestone, Cai-Yen et al. (2004) Genetically modified, live attenuated dengue virus type 3 vaccine candidates. Am J Trop Med Hyg 71:811-21
Murphy, Brian R; Blaney Jr, Joseph E; Whitehead, Stephen S (2004) Arguments for live flavivirus vaccines. Lancet 364:499-500
Whitehead, Stephen S; Hanley, Kathryn A; Blaney Jr, Joseph E et al. (2003) Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys. Vaccine 21:4307-16
Whitehead, Stephen S; Falgout, Barry; Hanley, Kathryn A et al. (2003) A live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys. J Virol 77:1653-7

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